IMPROVED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY/MASS SPECTROSCOPY (HPLC/MS) METHOD FOR DETECTION OF ANTHRAQUINONES AND ANTIOXIDANT POTENTIAL DETERMINATION IN ALOE SINKATANA 

Asim Halfawi Shargi¹, Mohammed Aboied², Ibrahim ME³,  Fatehalrahman F Magbool⁴*

¹Department of Pharmacogency, Pharmacy Program, Nile College, Khartoum , Sudan. Department of Pharmacogency, Faculty pharmacy, Sudan. International University,  Department of Pharmacogency, Pharmacy Program, Al Yarmouk College, Khartoum 

²Department of Chemistry, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan

³Al-Ribat National  University, Khartoum, Sudan, , Faculty of Pharmacy Khartoum, Sudan

⁴Department of Pharmaceutics, Khartoum University, P.O. Box 1998, Sudan

⁴Department of Pharmaceutics, Khartoum University, P.O. Box 1998, Sudan

ABSTRACT

Objectives:  Medicinal plants, either as an extract, pure compound or as a derivative, offer limitless opportunities for the discovery of new drugs. Sudan is a very rich source of medicinal plants which are used in the treatment of a wide range of diseases. Aloe sinkatana, has great potential to be developed as drug by pharmaceutical industries. The present study is undertaken to investigate the antioxidant potential of Aloe sinkatana by DPPH radical scavenging activity. In addition, the study also performed to explore the possibility of using HPLC-MS technique for the determination and analysis of Aloe sinkatana.

Methods:  The extracts of Aloe sinkatana were analyzed for antioxidant activity by using DPPH free radical scavenging activity. The results indicated that the extracts showed a high effective free radical scavenging in the DPPH assay, also these extracts exhibited a noticeable antioxidant effect at low concentrations. 

Results:  During in vitro evaluation the antioxidant potential of methanolic extract was the highest, followed by aqueous extract  in DPPH radical scavenging activity. So the methanolic extract of the plant, exhibited a great antioxidant effect at 50 μg/ml which may be attributed to high phenolic content. Therefore, methanolic extract to be a more active radical scavenger than aqueous extract. The HPLC-MS analysis had shown the methanolic extract of Aloe sinkatana to be rich in the major anthraquinones and their glucosides, which revealed 9 compounds, and also UV spectroscopy detected the presence of two flavonoids.

Conclusion:  The results indicated that the extracts of Aloe sinkatana  is a potential source of natural antioxidants or nutraceuticals with potential application to reduce oxidative stress with consequent health benefits. Due to stronger antioxidant potential and phytochemical composition, Aloe sinkatana could be proved as a valuable prospect in pharmaceutical formulations by taking part in the antioxidant defense system against generation of free radicals.

Keywords: Aloe sinkatana ,  anthraquinones , antioxidant, free radicals, HPLC/MS, medicinal plants.

INTRODUCTION

Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs¹. The study of plants used in traditional medicine requires the effective integration of information on chemical composition of extracts, pharmacological activities of isolated compounds, as well as indigenous knowledge of traditional healers. According to the World Health Organization, more than 80% of the world’s people depend on traditional medicine for their primary healthcare needs. The beneficial medicinal effects of these plant materials typically result from the combinations of secondary products present in the plant making the medicinal actions of plants unique to particular plant species or groups². Several studies have shown that plant derived antioxidant secondary metabolites scavenge free radicals and modulate oxidative stress-related degenerative effects^3,4. Free radicals have been beneficial in many diseases such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging^5,6. Previous research reports suggest that higher intake of antioxidant rich food is associated with decreased risk of degenerative diseases particularly cardiovascular diseases and cancer⁷.

Anthraquinones are a group of phenolics and are widely distributed in many plant families such as Fabaceae, Liliaceae, Rubiaceae and Rhamnaceae⁸. Anthraquinones derivatives such as emodine, physcione, rhein and chrysophanol have been used as colorants in food, drugs and cosmetics. Nowadays, anthraquinones attracted attention especially related to their interesting biological properties. They are known for their different biological activities including antimicrobial⁹, anticancer¹⁰, antioxidant¹¹ and anti-inflammatory¹². In this direction, it is suggested that consumption of anthraquinones-rich plants such as Rhamnus and Frangula species^13-15 can represent a valid intake way in order to benefit of their biological activities. From this point, the presence of anthraquinones is valuable as important criterion in the plants quality used for medicinal purposes. The recently revised family, Aloeacae, in the order Liliaceae, Liliflorae, was one of the widely distributed families of flowering plants. It consisted of 250 genera comprising 3700 species, mostly perennial herbs with rhizomes or bulbs¹⁶. When Dahlgren and Clifford (1982) made a major revision of superorders, orders and families within the monocotyledons, this family was sub-divided into several other families. Several free anthraquinones occur in roots and leaves of Aloe species. Aloe-emodin¹⁶  is a typical leaf constituent and is wide spread in the genus. Chrysophanol¹⁷ occurs both in roots and leaves^17,18, while aloe-emodin¹⁸ has been reported only from leaves¹⁹. Two main types of anthraquinones are present in Aloe, these are 1.8 dihydroxyanthraquinnone e.g chrysophanol and 7- hydroxyl aloe–emodin¹⁹. 

High-performance liquid chromatography (HPLC) is the usual technique for the determination of individual components in Aloe vera leaf extracts. There are already several examples of the use of HPLC for the analysis of aloin derivatives²⁰. All of them have been developed using a particulate reverse phase column. ElSohly et al.,²¹  have developed methods using both liquid chromatography/mass spectrometry (HPLC-MS) and liquid chromatography/photodiode array detection (HPLC/PDA) for the determination of aloe emodin and aloin A in aloe-based products. Rebecca et al.,²¹ also developed methods based on HPLC-MS for the studies of the exudates of the plants. However, only few reports are available on the antioxidant activity evaluation of Aloe sinkatana extract. So, the present study is undertaken to investigate the antioxidant potential of Aloe sinkatana by DPPH radical scavenging activity. In addition, the study also performed to explore the possibility of using HPLC-MS technique for the determination and analysis of Aloe sinkatana.

MATERIALS AND METHODS

Plant Material  

Leaves of Aloe sinkatana were collected in Erkawiet (East of sudan) in March 2009. They were kindly identified by Dr. Aalyia Awad, botanist, a voucher specimen was deposited under the registration  in the herbarium of the University of EL-Neelain. The leaves of the plant collected were separately dried at room temperature then finely ground with an electrical grinder.

Chemicals and Solvents

All chemicals and solvents used were HPLC grade. Laboratory and analytical grade chemicals, reagents, and solvents including; petrolum ether, chloroform, ethylacetate, butanol, methanol and 1,1-diphenyl-2-picrylhydrazyl (DPPH) , were from Merck India Ltd. 

Extraction and Fractionation

Leaves (50gm) were extracted for 3 hours by using soxhlet apparatus. The filtrate of this methanol extract was concentrated under reduced pressure until all the methanol had evaporated. The concentrate was redissolved in distilled water and lyophilized.

Determination of Antioxidant Activity (Scavenging Activity of DPPH Radical)

The DPPH free radical scavenging assay was carried out for the evaluation of the antioxidant activity. This assay measures the free radical scavenging capacity of the investigated extracts. DPPH is a molecule containing a stable free radical. In the presence of an antioxidant, which can donate an electron to DPPH, the purple colour typical for free DPPH radical decays, and the absorbance change at λ=517 nm is measured. This test provides information on the ability of a compound to donate a hydrogen atom, on the number of electrons a given molecule can donate, and on the mechanism of antioxidant action. The method was carried out as previously described by²². The methanolic and aqueous extracts were redissolved in methanol and 5% ethanol, respectively, and various concentrations (10, 50, 100, 500 and 1000 μg/ml) of each extract were used. Similar concentrations of ascorbic acid were used as positive control. The assay mixture contained in a total volume of 1 ml, 500 μl of the extract, 125 μl prepared DPPH (1 mM in methanol) and 375 μl solvent (methanol or 5% ethanol). After 30 min incubation at 25°C, the decrease in absorbance was measured at λ = 517 nm. The radical scavenging activity was calculated from the equation:

Where, %RSA=% of radical scavenging activity, Acontrol=Absorbance of control, Asample=Absorbance of sample

High Performance Liquid Chromatography HPLC/MS

The chromatographic apparatus consisted of a Model 616 pump, a Model 996 diode-array detector, a Model 717+ auto-sampler (all from Waters, Milford, MA, USA). An octadecyl silica glass cartridge column, Separon2 SGX C18 (150_3 mm I.D., 7 mm particle size) was used for the separation. The mobile phase contained 10% MeOH and 90% 5 mM aq. NH4OAc in water. For substituted anthraquinones, 40% MeOH±60% 5 mM aq. NH4OAc as the mobile phase was used, because in 10% MeOH±90% 5 mM aq. NH4OAc these compounds were strongly retained on the chromatographic column. The low rate for HPLC with UV detection was 1 ml/min. When the MS detection was applied, the low rate was reduced to 0.6 ml/min to enhance the electrospray response with optimum low rate after splitting. The post-column splitting 1:20 was used, so that 30 ml/min of eluent was introduced into the electrospray ion source. Samples were dissolved in the mobile phase and the injection volumes were 10 ml in all cases.

Figure 1: HPLC Spectrum of methanol extract of Aloe sinkatana leaf

RESULTS AND DISCUSSION

Various medicinal properties have been attributed to natural herbs. Extractive value useful for the evaluation of a crude drug and at the same time gives idea about the nature of the chemical constituents present, which is helpful for the estimation of specific constituents²³.

Medicinal plants constitute the main source of new pharmaceuticals and healthcare products. A whole range of plant derived dietary supplements, phytochemicals, and pro-vitamins that assist in maintaining good health and combating disease are now being described as functional foods and nutriceuticals. Plant- derived products are also increasingly accepted and used in the cosmetic industry. The roles of herbal plants in disease prevention and cure have been attributed, in part, to the antioxidant properties of their constituents of liposoluble and water soluble vitamins, and a wide range of amphipathic molecules, broadly termed phenolic compounds. 

Table 1: Antioxidant activity of different extract of Aloe sinkatana  leaf

The antioxidant effect of phenolic compounds is mainly due to their redox properties, and as a result of various possible mechanisms, which allow them to act as reducing agents, hydrogen donators, free radical scavengers, singlet oxygen quenchers, and/or metal chelators (transition- metal- chelating activity)²⁴. The extracts of Aloe sinkatana were analyzed for antioxidant activity by using DPPH free radical scavenging activity. The anthraquinone derivatives, such as aloe-emodin, emodin, rhein, chrysophanol, and physcion, are reported to possess antiangiogenic activity, by preventing blood vessel formation in zebra-fish embryos . Z.-H. He, M.-F. He, S.-C. Ma, and P. P.-H. But, “Anti-angiogenic effects of rhubarb and its anthraquinone derivatives,” Journal of Ethnopharmacology, vol. 121, no. 2, pp. 313–317, 2009. View at: Publisher SiteThe anticancer effect of aloe-emodin has been established in two human cancer cell lines, Hep G2 and Hep 3B. Aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines by different antiproliferative mechanisms. P.-L. Kuo, T.-C. Lin, and C.-C. Lin, “The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines,” Life Sciences, vol. 71, no. 16, pp. 1879–1892, 2002. View at: Publisher SiteIn our study all the extracts showed a high effective free radical scavenging in the DPPH assay (Table 1). These extracts exhibited a noticeable antioxidant effect at low concentrations. So the methanolic extract of the plant, exhibited a great antioxidant effect at 50 μg/ml (Table 1). The results were in agreement with those of R. Ashok Kumar, V. Rajkumar, and G. Guha, “Antioxidant and anti-cancer potentials of rheum emodi rhizome extracts,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 697986, 9 pages, 2011. View at: Publisher Swho found the methanolic extract to be a more active radical scavenger than aqueous extract. The HPLC analysis had shown the methanolic extract of Aloe sinkatana  to be rich in the major anthraquinones and their glucosides, which reveled 9 compounds, and also UV spectroscopy detected the presence of two flavonoids. Plant phenolic compounds especially flavonoids are currently of growing interest owning to their supposed properties in promoting health (anti-oxidants)²⁴. Flavonoids have been demonstrated to have anti-inflammatory, antiallergenic, anti-viral, anti-aging, and anti-carcinogenic activity²⁴. The broad therapeutic effects of flavonoids can be largely attributed to their antioxidant properties. In addition to an antioxidant effect, flavonoid compounds may exert protection against heart disease through the inhibition of cyclooxygenase and lipoxygenase activities in platelets and macrophages²⁴. Anthraquinones are structurally built from an anthracene ring (tricyclic aromatic) with a keto group each on carbon atom nine and ten. In plants, anthraquinones are found in a wide range of species¹¹. The effects of anthraquinones and anthrones are very diverse²⁴.  Anthraquinones and anthrones are very reactive and have a broad pharmacological activities including, potent anticancer, antidiabetic, antimicrobial, antiinflammatory, and cathartic properties as well as its cardio-, hepato-, and neuroprotective qualities. Anthraquinones and xanthones contain an aromatic core that serves as a scaffold for the attachment of diverse functional groups, resulting in a wide variety of molecules with distinct biological and biochemical characteristics²⁴.

CONCLUSION

Antioxidant-rich plant extracts serves as sources of nutraceuticals that alleviate the oxidative stress and therefore prevent or slow down the degenerative diseases. An effort has been made to explore the antioxidant properties of commercial available herbal extracts. Our results show that medicinal plants can be promising sources of natural products with potential antioxidant activity. The results indicated that the extracts of  Aloe sinkatana  is a potential source of natural antioxidants or nutraceuticals with potential application to reduce oxidative stress with consequent health benefits.

ACKNOWLEDGEMENTS 

The authors are thankful to wish to Department of Pharmacogency, Faculty of Pharmacy, Nile University,  Khartoum, Sudan. 

COMPETING INTERESTS 

The authors declare that they have no competing interests.

AUTHOR’S CONTRIBUTION

The manuscript was carried out, written, and approved in collaboration with all authors.

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